Nucleic acid hybridization has found widespread application in genetic research, biomedical research and clinical diagnostics. The development of a technology for "mixed phase hybridization" (hybridizing probes to immobilized target DNA) and for cloning of unique sequence genetic probes has lead to major advances in basic and applied areas of biochemistry and medicine (For a review see: Meinhoth, J. and Wahl, G., (1984) Analytical Biochemistry 138, 267-284). In the standard hybridization reaction, a radioisotope-labeled probe is annealed to a DNA or RNA sample which has been immobilized on an inert solid support. The detection of a radioactive signal by autoradiography indicates the presence or absence of the complementary nucleic acid sequence in the target sample. In the past, the potential health hazards, disposal problems and instability of radionucleotides have placed limitations on the use of nucleic acid probes. This has lead to great interest in the development of alternative DNA labeling and detection systems which do not contain the drawbacks inherent in the use of radioisotopes.
The development of a nonradioactive DNA detection system requires a method to label the nucleic acid probe and a means to recognize and detect the annealed probe-target hybrid. The high binding constant and specificity of biotin for avidin or streptavidin (reviewed in Green, M., (1975) Advances in Protein Chemistry, 29, 85-133) have been used in different systems to localize and detect a wide range of target molecules.
The principle of the biotin-avidin interaction as the basis of probe-ligand interactions in molecular biology was reviewed by Bayer and Wilchek in 1979: "Thus (in addition to biotin-requiring enzymes) biotin-derivatized hormones, phages, lectins, antibodies and other binding proteins can interact with avidin; and if the avidin is immobilized or covalently bound to a potentially perceptible probe, the avidin-biotin complex can be used for the localization or isolation of the compounds above and/or their receptors" (Bayer, E. A. and Wilchek, M. (1979), Methods in Biochemical Analysis 26, 1-45). This principle is further described in U.S. Pat. No. 4,228,237 (Hevey, R. C. and Malmros, M. K., Oct. 14, 1980) in wnich an avidin-coupled signaling enzyme is used to recognize a biotin-labeled reagent which will specifically bind to the ligand in question.
Other examples of biotin-avidin interactions as applied to probes are contained within an article by Langer et al. (Langer, P. R., Waldrop, A. A. and Ward, D. C. (1981) Proc. Natl. Acad. Sci., USA 78 6633-6637), who describe the synthesis of biotin-UTP and biotin-dUTP analogs which are substrates for a variety of RNA and DNA polymerases. The disclosures in this publication were further set forth in European Patent Application EP No. A200063879, in which it is claimed that biotin may be covalently linked to the 8-position of a purine base or the 5-position of a pyrimidine base in a nucleoside triphosphate.
However, it is stated by Ward, et al., in that application that " . . . probe moieties should not be placed on ring positions that sterically, or otherwise, interfere with the normal Watson-Crick hydrogen bonding potential of the bases. Otherwise, the substitutents will yield compounds that are inactive as polymerase substrates . . . Normally, such considerations limit substitution positions to the 5-position of a pyrimidine and the 7-position of a purine or a 7-deazapurine." (The Ward disclosure and claims show that the 8-position of a purine was intended.) This statement reflects the common supposition that if labels are attached to nucleotides at the hydrogen bonding positions the modified nucleotides cannot be useful in probes.
Notwithstanding the teachings of Ward and others, the inventors herein have discovered that nucleotide analogs in which biotin is attached at the hydrogen bonding position of the purine or pyrimidine base may be iccorporated into probes. Moreover, probes labelled in this manner will still hybridize to "target" DNA in highly sensitive detection protocols. This result has occasioned the production of a new class of labeled nucleotides, the usefulness of which was hitherto unsuspected.